Primer generation-rolling circle amplification method optimized for the detection of pathogenic bacteria

Abstract

With advancements in DNA amplification research, isothermal amplification technology has emerged as an attractive method for detecting target DNA. Here, we describe primer generation-rolling circle amplification (PG-RCA) as an isothermal amplification method for detecting Escherichia coli O157:H7, Salmonella Typhimurium, Bacillus cereus, and Listeria monocytogenes. To improve PG-RCA sensitivity, the concentrations of the reaction components, dNTPs, phi29 DNA polymerase, and circular probes were optimized; the optimized conditions were applied to detect each target bacterium. A pair of forward and reverse circular probes that hybridized to the sense and anti-sense target genes was used in PG-RCA, exhibiting target selectivity. PG-RCA, which generated additional primers simultaneously with linear RCA and comprised multiple reaction cycles, resulted in higher accumulation of amplified DNA products than did linear RCA within the same reaction period. The threshold time (Tt) for each target gene concentration was determined based on the threshold value set in the amplification plot for PG-RCA, and a linear correlation between the Tt value and genomic DNA concentration was proven for each of the four bacteria. The PG-RCA-based assay could be applied to gene-based detection of various microorganisms and may be a useful isothermal amplification method for replacing traditional PCR methods.